Ntoukakis, V. | bioRxiv Science

bioRxiv2021

Expression of putative effectors of different Xylella fastidiosa subspecies/strains reveals recognition and defense activation in various model plants

The re-emergence of Gram-negative bacterium Xylella fastidiosa in Europe in 2013 impelled the scientific community to discover novel strategies for crop protection. The wide host range of Xylella indicates the existence of yet not characterized pathogenic mechanisms to overcome plant defenses. The recent uprising accuracy of a variety of bioinformatics tools, with the ability to predict the function of putative microbial protein represent a useful approach for understanding which of these proteins are associated with pathogens virulence. In this study we collected a number of putative effectors from two X. fastidiosa strains: Temecula1 and CoDiRo and the subspecies (ssp.) Sandyi Ann-1. We designed an in-planta Agrobacterium based expression system that drives the expressed proteins to the cell apoplast, in order to investigate their ability to activate defense in various model plants. Furthermore, we organized the resulted proteins according to their sequential and structural similarities via the I-TASSER online tool. We identified that various X. fastidiosa proteins were able to differentially elicit cell death-like phenotypes in Nicotiana tabacum, N. sylvestris and N. benthamiana. These proteins are members of different enzymatic groups: a) hydrolases/hydrolases inhibitors, b) serine proteases and c) metal transferases. Collectively, we identified structurally similar proteins that were able to differentially elicit cell death-like phenotypes in different cultivars of the same species. Our findings provide the bases for further studies on the mechanisms that underlie host-defense activation by X. fastidiosa putative effectors, as well as, pathogens adaptation in susceptible hosts.

microbiology↗

bioRxiv2021

ACTIVATION LOOP PHOSPHORYLATION OF A NON-RD RECEPTOR KINASE INITIATES PLANT INNATE IMMUNE SIGNALING

Receptor kinases (RKs) play fundamental roles in extracellular sensing to regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) function primarily as peptide receptors that regulate myriad aspects of plant development and response to external stimuli. Extensive phosphorylation of LRR-RK cytoplasmic domains is among the earliest detectable responses following ligand perception, and reciprocal transphosphorylation between a receptor and its co-receptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model RK, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in anti-bacterial immunity. These results reveal a non-catalytic role for the EFR cytoplasmic domain in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RK complexes with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor which could initiate signaling in a feed-forward fashion either allosterically or through driving the dissociation of negative regulators of the complex.

biochemistry↗